Leptin, GABAergic Neurons and Feedback / Feedforward Regulation of Energy Balance Neurons in the arcuate nucleus (ARC), especially AgRP and POMC neurons, play key roles in regulating energy balance. They receive feedback information on the current status of fat stores (via leptin) and ?feedforward? information on future, anticipated changes in energy balance. The neural mechanisms responsible for feedback and feedforward regulation, however, are unknown. Previous studies have suggested an important role for GABAergic neurons; in the ARC in relaying feedback regulation, and in the dorsomedial hypothalamus (DMH) in relaying feedforward regulation. With regards to feedback regulation, progress has been hampered by lack of knowledge regarding ?unknown? neurons that reside in the ARC. In short, we have lacked a ?parts list?. Recently, using single neuron transcriptomics, we discovered many novel GABAergic neurons in the ARC, including some which express LEPRs (Campbell JN et al., Nat Neurosci, 2017). Building on these discoveries, Aims 1 and 2 will address mechanisms of feedback regulation. Aim 3 will extend our discovery that vDMH GABAergic neurons mediate feedforward regulation of AgRP neurons. Aim 1: To use single neuron transcriptomics to assess leptin?s effects on gene expression in all subtypes of ARCLepr and vDMHLepr neurons. By capturing nuclei from all LEPR-expressing neurons in the ARC and vDMH, and looking at genome-wide transcriptional responses to leptin at the single nuclei level, this Aim will identify cell type-specific transcriptional effects of leptin in all subtypes of LEPR-expressing neurons. Aim 2: To investigate the role of novel ARCGABAergic neurons in regulating energy balance. The following neurons will be studied: LEPR-expressing, GABAergic ARCTrh/Cxcl12 neurons ? which express the highest level of LEPRs in the ARC, LEPR-expressing, GABAergic ARCTbx19 neurons ? which are transcriptionally similar to the leptin-regulated ?RIP-Cre? neurons that control energy expenditure, GABAergic ARCTrh-Lef1 neurons ? which are enriched for transcripts corresponding to human body mass index (BMI) GWAS-linked loci, GABAergic ARCSST neurons ? which are transcriptionally similar to AgRP neurons and thus likely to share functions, and finally GABAergic ARCHtr3b neurons ? which are transcriptionally distinct but express the interesting, unique ionotropic serotonin receptor, 5-HT3AB. Aim 3: To determine the means by which food-cue, ?feedforward? regulation reaches the vDMHLepr- GABAergic AgRP circuit. We and others unexpectedly found that AgRP neurons, in addition to being regulated by feedback signals such as leptin, are also regulated rapidly by sensory cues that ?anticipate? food consumption. Importantly, this is mediated, at least in part, by vDMHLepr-GABAergic neurons which synapse onto AgRP neurons. This Aim seeks the source of this regulation. Our studies suggest a key role for a functionally distinct subset of glutamatergic neurons in the lateral hypothalamus.